1. Field of the Invention
The present invention relates to an imaging of a matrix disposed on a surface of a biochip, which includes a substrate and a plurality of biological materials disposed on a surface of the substrate in a matrix form, and also relates to an analysis of the components of the matrix.
2. Description of the Related Art
A biochip, such as a DNA chip, protein chip and so on, which includes a substrate and various molecular probes disposed on a surface of the substrate in a matrix form, has been employed for the purposes of analyzing a genome or analyzing a generation of a gene. Further, it is expected that the result of the analysis employing biochips provides a critical index for diagnosis of cancers, genetic diseases, life style-related diseases, infectious diseases and the like, prediction for prognostics, or a decision on treatment policy and so on.
Several methods for preparing biochips are known. Exemplary methods for preparing a DNA chip may include: a method of consecutively synthesizing DNA probes directly onto a substrate by using photolithography (U.S. Pat. No. 5,405,783 and so on); or a method for supplying synthesized DNA or synthesized cDNA (complementary DNA) onto a substrate and binding it thereto (U.S. Pat. No. 5,601,980, Japanese Patent Laid-Open No H11-187,900 (1999), an article in “SCIENCE”, Vol. 270, pp. 467 (1995) and so on).
In general, the biochip is formed by using one of the two methods described above, and when the thus-formed biochip is used for the applications described above, it is critical to know quantities, i.e., densities, of biological materials used for forming probes that are included in matrices for the purpose of ensuring the credibility of the analysis, i.e., the quantification or the reproducibility of the analysis. Further, it is also critical to know what type of matrix dimension (i.e., shape, size or condition) is provided to the matrix existing thereon (i.e., imaging) for the purpose of assuring the quantification-ability or the reproducibility of the analysis. In addition, as described later, if there is no physical address for indicating the expected position of a matrix on the substrate that is employed for forming chips, an additional problem may occur. More specifically, when the biochip is formed by using a method of supplying fine droplets of a probe solution thereto via the ink jet method, for example, an absence of the physical address thereon may lead to an unclear determination of the position of the probe portion when the analysis is conducted on the biochip, depending on employed method. In such case, the detection means itself must also enable a clear determination of the matrix position.
However, the probe on the biochip exists principally as a monolayer or less, and in general, the analysis of the biological materials including the clear determination of the matrix position requires highly sensitive surface analysis techniques.
One of the known highly sensitive surface analysis techniques that satisfies the aforementioned requirements may be a method of using stable isotope labeled probes. However, this method has various disadvantages from the viewpoint of general purpose usage. Specifically the method requires complicated labeling, as well as special facilities and special equipment, because the employed isotope itself may be a source of a radioactive emission.
Another method may be that of labeling the probe with a fluorescent label, or alternatively, that of labeling a specific material that specifically binds to the probe with a fluorescent label and then binding it to the probe, which is known as a fluorescent-hybridization method for the DNA chip. However, such a method also has various problems with respect to achieving higher quantification-ability, such as a problem of the chemical stability of the fluorescent dye used for labeling, a problem of the fluorescent quenching, a problem of the nonspecific adsorption of the fluorescent dye onto the substrate surface, or additionally the problem of the quantification-ability (i.e., stability, reproducibility) of the specific binding-ability (i.e., hybridization) Thus, there are a number of problems for quantitatively detecting the amount of the existing probe itself.
Other highly sensitive surface analysis methods that are capable of being employed for analyzing general detection objects include the ATR method that utilizes FT-IR (Fourier Transform Infra Red Spectroscopy), XPS (X-ray Photoelectron Spectroscopy) and so on. However, these methods do not involve sufficient sensitivity for the quantitative analysis of the probe on a biochip, i.e., a biological material, or imaging thereof. In particular, when a general purpose glass is employed as a substrate for producing the biochip, these methods are not available, because the absorption due to the glass substrate itself adversely affects the analysis results when the FT-IR (ATR) method is employed, for example, or because a charge-up occurred on the glass, which is an electrically insulating material, adversely affecting the analysis when the XPS method is employed.
Yet another highly sensitive surface analysis method that is capable of being employed for analyzing biological related materials may be a DNA detection method utilizing the laser RIS (Resonance Ionization Spectroscopy) method, which is disclosed in U.S. Pat. No. 5,821,060. In this method, the specimen surface is irradiated with laser or ion beams mentioned below, and the generated portion is irradiated with a laser beam having a wavelength that is equivalent to ionization energy of a specific element, so that the specific element is ionized and emitted from the specimen surface and the emitted ionized element is detected. Disclosed methods for releasing the element from the specimen surface may be a method utilizing a laser beam (laser ablation) or a method utilizing ions (ion sputtering). However, these methods have a technical limitation in that only a limited number of elements can be detected.
Yet another highly sensitive surface analysis method may be dynamic SIMS (Secondary Ion Mass Spectrometry), in which an organic compound is decomposed to smaller fragment ions or to particles during the process of generating a secondary ion. Thus, the amount of the information on the chemical structures obtained from the mass spectrum is not sufficient. Thus, the method is not generally suitable, because the obtained information is not sufficient for the analysis of organic compounds such as, for example, nucleic acid-related materials having only four common bases.
On the other hand, the time of flight secondary ion mass spectrometry (TOF-SIMS), which is also known as another technique of the secondary ion mass spectrometry, is an analysis method for investigating what types of atoms or molecules exist on the uppermost surface of a solid specimen. This method has the following advantages: having an ability to detect a trace amount of a component of 109 atoms/cm2 (equivalent to 1/105 of all atoms existing in one atomic layer of the uppermost surface); being applicable to both organic and inorganic compounds; being capable of detecting all types of elements and compounds that exist on the surface; and being able to image secondary ions from materials that are on the surface of the specimen.
Here, the principles of the time of flight secondary ion mass spectrometry will be described as follows.
At high vacuum, a high speed pulsed ion beam (primary ion) irradiated to a surface of a solid specimen causes sputtering, in which a structural component of the surface is emitted into the vacuum. Ions (secondary ions) having positive or negative charges generated during this process are accelerated into a mass spectrometer, where they are mass-analyzed by measuring the travel time from the specimen surface to a detector. In the sputtering process, various ions having a variety of masses are generated depending on the chemical components of the surface of the specimen, and the ions having a smaller mass fly faster and, on the contrary, ions having a larger mass fly slower, within a constant electrical field. Thus, detecting the time elapsed from the generation of the secondary ions to the arrival of the generated ions to the detector (i.e., time of flight) provides an analysis of the mass of the generated secondary ions.
On the other hand, in the dynamic-SIMS method, organic compounds are decomposed to small fragment ions or particles during the ionization process as stated above. Thus, information on the chemical structure obtained from the mass spectrum, e.g., mass range, is limited. On the contrary, in the TOF-SIMS method, the structures of the organic compounds can be directly obtainable from the mass spectrum with a wide mass range, because a much smaller amount of the primary ions is necessary in the TOF-SIMS method, so that while the organic compounds are ionized, they substantially retain their chemical structure. In addition, the information on the uppermost layer (within a depth of several angstroms) of the object can be selectively obtained as only the secondary ions generated in the uppermost solid surface are emitted into the vacuum.
The TOF-SIMS apparatus that employs the principle of the measurements described above is generally classified as a sector-type apparatus and a reflectron-type apparatus. One of the differences between these two types is in the manner of electrically grounding of a holder that fixes an object to be analyzed. In the sector-type apparatus, the generated ions are led to the mass spectrometer by applying positive or negative voltage of several kV to the specimen-fixing holder. In the reflectron-type apparatus, the specimen-fixing holder is grounded and the secondary ions are led to the mass spectrometer by applying positive or negative voltage of several kV to several-ten kV to an extracting electrode for the secondary ions.
The TOF-SIMS method often utilizes positive primary ions, and both positive secondary ions and negative secondary ions are generated regardless of the polarity of the utilized primary ions. Also, regardless of the polarity of the utilized primary ions, the amount of the secondary electrons that are generated by irradiating the primary ions is greater than the primary ions under the general measurement conditions, so that the surface potential tends to be positive. In turn, when the positive charge accumulates beyond a certain level (i.e., charge-up condition), the excessive positive charge may disturb the quantitative measurements. In considering the apparatus configurations in relation with the charge-up condition, the measurements of the negative secondary ions from the insulator material by using the sector-type apparatus can cause the highest positive-charge accumulation, because all of the generated secondary electrons are directed toward the extracting electrode for the (negative) secondary ions, wherein the above-mentioned positive voltage is applied to the extracting electrode.
In order to neutralize the positive charge caused by the above-mentioned charge-up condition, both the sector-type apparatus and the reflectron-type apparatus may often be equipped with a pulse-type electron gun for neutralizing the charge. A specific method for neutralizing the charge by using the pulse-type electron gun may include a step of applying the electron beam from the above-mentioned pulse-type electron gun onto the object to be analyzed for a constant duration irradiating primary ions (sub-nanosecond pulse to several nanosecond pulse) and before irradiating the primary ions for the next process of generating secondary ions. Here, while the electron beam is irradiated by the pulse-type electron gun onto the object to be analyzed, the application of the voltage to the object holder (for the sector-type apparatus) or to the secondary ion extracting electrode (for the reflectron-type apparatus) is stopped, and the holder or the electrode is grounded, respectively.
The above-mentioned method of neutralizing the charge often relieves (or compensates for) the accumulated positive charge, enabling the analysis of the insulator material. Here, when the negative secondary ions are measured for the insulator material by using the sector-type apparatus, the insulator is most-considerably and positively charged, and thus the margin of the charge-neutralization in this type of measurement is the narrowest. In order to prevent the charge-up, using the reflectron-type apparatus, in which the object holder is constantly electrically grounded, is (in general) more advantageous than using the sector-type apparatus. In particular, when the object to be analyzed has a lower electric conductivity (in other words, higher electric resistivity or a lower dielectric constant), e.g., glass and the like, a reflectron-type apparatus is more suitable for carrying out the quantitative measurements.
Regardless of whether a reflectron-type apparatus or a sector-type apparatus is employed, the TOF-SIMS method is the analysis method of a considerably higher sensitivity. This method enables the analysis of an object and is less influenced by a charge-up, e.g., oligonucleotide formed in a single molecular film level on a gold substrate having better electric conductivity. (Proceeding of the 12th International Conference on Secondary Ion Mass Spectrometry, 951 (1999)). Further, an evaluation conducted by the present inventors shows that, by conducting the process of preventing the charging-up, biological materials such as oligonucleotide bound to the substrate surface with a higher dielectric constant, such as a glass substrate, can be in-situ analyzed by irradiating the primary ions at a spot several μm in diameter when the analysis is conducted by an individual spot measurement.
However, the evaluation conducted by the present inventors also shows that when the two-dimensional secondary ion image was to be obtained by sequentially scanning the primary ion beam having a beam diameter of 5 μm in a constant direction, like the scanning line of a TV receiver (i.e., raster scanning), onto the substrate of a higher resistivity across a wide area, e.g., the area that is 500 μm×500 μm, a good image was not obtained because of considerable influence of the charge-up.